Intestinal Dominance by Multidrug-Resistant Bacteria in Pediatric Liver Transplant Patients

ABSTRACT Pediatric liver transplantation (PLTx) is commonly associated with extensive antibiotic treatments that can produce gut microbiome alterations and open the way to dominance by multidrug-resistant organisms (MDROs). In this study, the relationship between intestinal Relative Loads (RLs) of β-lactamase genes, antibiotic consumption, microbiome disruption, and the extraintestinal dissemination of MDROs among PLTx patients is investigated. 28 PLTx patients were included, from whom 169 rectal swabs were collected. Total DNA was extracted and blaCTX-M-1-Family, blaOXA-1, blaOXA-48, and blaVIM were quantified via quantitative polymerase chain reaction (qPCR) and normalized to the total bacterial load (16SrRNA) through LogΔΔCt to determine the RLs. 16SrRNA sequencing was performed for 18 samples, and metagenomic sequencing was performed for 2. Patients’ clinical data were retrieved from the hospital’s database. At least one of the genes tested were detected in all of the patients. The RLs for blaCTX-M-1-Family, blaOXA-1, blaOXA-48, and blaVIM were higher than 1% of the total bacterial population in 67 (80.73%), 56 (78.87%), 57 (77.03%) and 39 (61.9%) samples, respectively. High RLs for blaCTX-M-1-Family, blaOXA-1, and/or blaOXA-48, were positively associated with the consumption of carbapenems with trimethoprim-sulfamethoxazole and coincided with low diversity in the gut microbiome. Low RLs were associated with the consumption of noncarbapenem β-lactams with aminoglycosides (P < 0.05). Extraintestinal isolates harboring the same gene(s) as those detected intraintestinally were found in 18 samples, and the RLs of the respective swabs were high. We demonstrated a relationship between the consumption of carbapenems with trimethoprim-sulfamethoxazole, intestinal dominance by MDROs and extraintestinal spread of these organisms among PLTx patients. IMPORTANCE In this study, we track the relative intestinal loads of antibiotic resistance genes among pediatric liver transplant patients and determine the relationship between this load, antibiotic consumption, and infections caused by antibiotic-resistant organisms. We demonstrate that the consumption of broad spectrum antibiotics increase this load and decrease the gut microbial diversity among these patients. Moreover, the high loads of resistance genes were related to the extraintestinal spread of multidrug-resistant organisms. Together, our data show that the tracking of the relative intestinal loads of antibiotic resistance genes can be used as a biomarker that has the potential to stop the extraintestinal spread of antibiotic-resistant bacteria via the measurement of the intestinal dominance of these organisms, thereby allowing for the application of preventive measures.

interpretation for the reader. While very interesting findings are buried in the text, every sub-section of results contains too much detail obscuring main points. There are 16 pages of results (not including supplementary materials), which is quite long for this study. Many tables include an exhaustive breakdown of each patient, when more of a summary presentation should be presented. By including this degree of detail, the authors are leaving too much of the interpretation work to the reader, making it challenging to complete reading and draw conclusions. I think several of the tables and figures could be transformed into violin plots (or similar) with an axis being time (before vs after a specific antibiotic, or transplant, etc). I also think the text should be decreased by at least 25%, evenly throughout the results section. 2. The authors conclude their introduction with "our aim is to evaluate the potential benefit of tracking RLs of these genes in the gut microbiome as a biomarker for personalized medicine approaches in managing pediatric transplant patients", but that is not what the manuscript accomplishes. The manuscript is more of a descriptive piece documenting the incidence and natural history of MDR genes. Perhaps the authors mean that this aim is the next step in this work, to evaluate how these gene RLs could be used for managing pediatric transplant patients. 3. Important details are missing from methods section 2.1. Where these patients inpatients every time they were sampled? Why were some only sampled a few times but others sampled many times? It is also not clear until much later in the manuscript (and still even not entirely clear) what "extra-intestinal isolates" are. Also, later in the manuscript group 1 and group 2 appear without explanation. Section 2.1 should better explain how patients were selected for this cohort, how they were sampled, how long they were followed, and how they were grouped. 4. The statistical analysis section is vague and does not orient the reader to what the primary analysis is going to be. It foreshadows a very descriptive manuscript. 5. While not possible to change, a major limitation of this study is the lack of pre-transplant data points. If one of the conclusions is that antibiotics leads to increased abundance of MDROs, how can we conclude that without data points prior to the initial antibiotic load (i.e. transplant)?
Minor feedback: -Is "dominance" the right term? Catchy and powerful, but what is the definition of this? -Does the presence of these genes equate to drug resistance? In other words, does the presence of these genes ensure the presence of organisms with drug resistance? -In 2.6 it was not entirely clear to me what "P1" was until I later saw a table listing every subject as P#. This study focused on the relationship between intestinal loads of antibiotic resistance genes specifically beta-lactamase among pediatric liver transplantation patients. The concept of the study is interesting; the study is well designed and written in good English. Introduction explained the importance and aim of study clearly. The material and method and result sections are well being explained. However, some points need to be clarified for me. Why the authors used 18 samples from one patient? Are the results obtained from only one patient accurate? Was this P1 patient breastfeed? If yes, do you checked the mother in terms of antibiotic consumption? Why didn't the authors use, for example, 5 samples at different time points from more than one patient? Staff Comments:

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the information as necessary. Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER. • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file. • Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file. For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process. Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript. " Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail. Arrangements for payment must be made before your article is published. For a complete list of Publication Fees, including supplemental material costs, please visit our website.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees. Need to upgrade your membership level? Please contact Customer Service at Service@asmusa.org.
Thank you for submitting your paper to Microbiology Spectrum.

Dear Reviewers,
We would like to take this chance to thank you for your constructive criticism of our manuscript and your helpful insight regarding how to improve it. We have addressed all the points raised in your reports and what follows is a point-by-point breakdown of the changes done in the manuscript:

Reviewer #1:
Reviewer: My primary concern about the manuscript is that a lot of granular data is presented without enough refinement and interpretation for the reader. While very interesting findings are buried in the text, every sub-section of results contains too much detail obscuring main points. There are 16 pages of results (not including supplementary materials), which is quite long for this study. Many tables include an exhaustive breakdown of each patient, when more of a summary presentation should be presented. By including this degree of detail, the authors are leaving too much of the interpretation work to the reader, making it challenging to complete reading and draw conclusions. I think several of the tables and figures could be transformed into violin plots (or similar) with an axis being time (before vs after a specific antibiotic, or transplant, etc). I also think the text should be decreased by at least 25%, evenly throughout the results section.

Authors:
The results section has been extensively modified. Mainly, the following key changes were made: -The text has been greatly reduced and only the most important findings were stated in the text. It was not always possible to highlight these changes because a great part of these modifications were deleting the text and adding the information to tables.
-The tables were collapsed into much simpler ones where the summary of the data is presented. The table titles were highlighted to indicate that the entire table has been modified.
-Regarding the violin plots, we made several attempts to transform some of the data into them but since there were very little points in the "before" category (i.e. before transplantation or antibiotic consumption, etc…) the graphs were not very representative of the data. We instead summarized most of the data, reduced the level of individual details, and presented them in a more global view of the study.
-Sections 3.2 and 3.3 were merged (and so the numbering of the subsequent sections were changed) and a lot of the text was removed and transformed into tables.
-The information in Table 4 in what is now section 3.2 (previously section 3.3) was found to be redundant and therefore the table was removed.
-In what is now section 3.4 (previously 3.5), Tables 5 and 6 were removed and added to Supplementary Tables 1 and 2 since the important and significant associations were mentioned in the text and the tables seemed redundant.
-In section 3.6 (previously 3.7), no significant reduction of the text was made since we believe that the associated Figure is highly complex and would be difficult to understand without the detailed information given in the text. Various attempts were made and in all of them, the information after reduction seemed lacking.
We hope that by these changes the presentation of the results became clearer and more informative.

Reviewer:
The authors conclude their introduction with "our aim is to evaluate the potential benefit of tracking RLs of these genes in the gut microbiome as a biomarker for personalized medicine approaches in managing pediatric transplant patients", but that is not what the manuscript accomplishes. The manuscript is more of a descriptive piece documenting the incidence and natural history of MDR genes. Perhaps the authors mean that this aim is the next step in this work, to evaluate how these gene RLs could be used for managing pediatric transplant patients.

Authors:
The authors agree with this point and therefore this sentence was removed from the introduction since it is an "overreaching aim" for potential future applications.
Reviewer: Important details are missing from methods section 2.1. Where these patients inpatients every time they were sampled? Why were some only sampled a few times but others sampled many times? It is also not clear until much later in the manuscript (and still even not entirely clear) what "extra-intestinal isolates" are. Also, later in the manuscript group 1 and group 2 appear without explanation. Section 2.1 should better explain how patients were selected for this cohort, how they were sampled, how long they were followed, and how they were grouped.
Authors: Methods Section 2.1 was updated in order to clarify how the patients were selected, how the samples were obtained, how long the patients were followed, how they are grouped, and the nomenclature used. The term "extra-intestinal isolates" was also defined in this section. All the changes are highlighted in the marked-up manuscript. Moreover, Table 2 in the Results Section 3.1 was heavily modified in order to give a better global image regarding the patients and samples included in this study (including an update regarding from which wards the samples were received) and will hopefully answer all the questions raised in this point.

Reviewer:
The statistical analysis section is vague and does not orient the reader to what the primary analysis is going to be. It foreshadows a very descriptive manuscript.